This invention relates generally to a method for sphering or sphering and fixing whole blood erythrocytes without volume change for accurate and precise cell volume measurement. More particularly, the method employs a series of dilution steps whereby a protein, externally provided or endogenously supplied and a sphering agent are added in a protein/sphering agent weight ratio of from about 20:1 to about 70:1, based on total sample volume, and the concentration of detergent in the final sample is from about 2 mg./100 ml. to about 10 mg./100 ml.
Methods which utilize the measured amount of light scattered from individual red cells (erythrocytes) to determine the individual and mean volumes of red cells, suffer from two kinds of error:
1. The native human red cell is a biconcave disc and the amount of light scattered within a particular solid angle varies with the orientation of the cell with respect to the incident light beam; PA1 2. During handling, i.e. dilution and pumping, the shape of the cells can change depending in part on the time between the drawing of the blood and the time of measurement and in part on the composition of the diluted blood sample.
For a discussion of the above, see Hemolysis and Related Phenomena, Chapter II, pp 10-49 by Eric Ponder (1948) and Transformation and Restoration of Biconcave Shape of Human Erythrocytes Induced by Amphiphilic Agents and Changes of Ionic Environment, Biochemica Et. Biophy. Acta, Bernard Deuticke, pp 494-500 (1968).
The present invention eliminates both of these sources of error and permits vastly improved methods for determination of human red blood cell volumes. It is well known, see for example Ponder supra, that it is possible to sphere red blood cells in isotonic solution without changing their volumes. Since the light scattering from a perfectly sphered cell is invariant with orientation in a light beam, the first kind of error is eliminated. However, such preparations are notoriously unstable and red cell lysis occurs at various times after sphering, depending on the choice of sphering agent and the properties of the individual blood samples.
It has now been discovered that prolonged stability of the sphered state can be achieved by controlling the absolute concentration of the sphering agent (typically a material with detergent properties) and the weight ratio of sphering agent to protein, either added or endogenous at any desired dilution in isotonic solution. This helps to assure shape consistency during processing and minimizes the second kind of error.
The method of this invention can be carried out generally in two ways:
A. A blood serum sample is diluted, typically about 1/1000, in an isotonic solution containing sphering agent (detergent) and albumin at the required concentrations; or
B. The blood serum sample is diluted with an amount of isotonic solution containing the sphering agent at a concentration which is just sufficient to cause sphering when the dilution provides the correct ratio of sphering agent to the endogenous serum albumin (plasma protein) from the blood sample itself. The resulting sample is then simultaneously and/or successively fixed and further diluted by adding an isotonic solution of a fixing agent to harden the sphered cells and make them completely insensitive to processes which could otherwise cause them to change their shape or size or lyse and lose their contained hemoglobin.